I. Clinical and Genomic Studies of Uveal Coloboma I have recruited and examined 146 families (562 participants) where at least one member is affected by uveal coloboma. All probands and their first-degree relatives (when available) have complete ophthalmic exams. General physical examinations and targeted systemic testing (e.g., renal ultrasounds, echocardiograms) were performed on probands, as needed. Lymphoblastoid cell lines were established on all participants. To date, we have analyzed 217 study subjects from 66 families using a custom-capture, high-throughput sequencing platform (Illumina HiSeq2000). The 196 known genes on the platform include: established coloboma genes, candidate genes from our unbiased screen of laser-capture microdissected (LCM) developing mouse tissue (see also below), and genes known to cause coloboma in animal models. Although the yield on molecular testing for known genes in this largely non-syndromic coloboma cohort is <5%, we identified several candidates. Among others, variants in BMP7 and RARB (missense), TFAP2A (frameshift) and CHD7 (nonsense) were identified in 4 unrelated families and confirmed by Sanger sequencing. All variants had not been previously reported, were predicted to be deleterious and segregated in the affected individuals. The RARB mutation is located in the conserved DNA binding domain and predicted to result in a single aminoacid substitution. Morpholino knockdown in zebrafish embryos of the ortholog rarga gene resulted in a coloboma phenotype. As part of our whole exome/whole genome protocol, we were awarded a competitive grant for sequencing 75 exomes through the NIH Clinical Center Genomics Opportunity. We have enrolled a total of 58 participants with largely syndromic coloboma and other developmental eye defects. Exome sequencing is being performed on all new families, and existing families for whom we can obtain appropriate consent. II. Laboratory Studies of Uveal Coloboma A. Mouse Models of Coloboma. 1. A novel Pax2 mutant mouse My lab had published a mouse model of autosomal dominant congenital optic nerve excavation caused by a missense mutation (p.T74A) in the paired domain of the Pax2 gene. Pax2 is dynamically expressed at the closing edges of the optic fissure and homozygous mutation results in uveal coloboma. The Pax2 mutant mouse is an ideal model to study the role in optic fissure closure of the transcriptional pathway regulated by Pax2. We performed differential profiling experiments in developing Pax2 mutant embryos (heterozygous and homozygous) compared to wild type across three developmental time points for optic fissure closure (E10.5, E11.5, and E12.5). A shortlist of 83 candidate genes differentially expressed across the three timepoints was prioritized based on expression (eye and ventral optic fissure), association with disease in human, available studies in animal models, eye related conditions, and known function. Of the 20 top ranking genes, those containing putative Pax2 binding sites in their promoter region (direct targets) were selected for further expression and functional studies in mouse and zebrafish. 2. The RICO mouse The RICO mouse arose from the random insertion of a transgene (NSE-VEGF) on chromosome 13 in the C57BL/6 background. Both homozygous and heterozygous mutants develop coloboma. We have extensively characterized the genomic organization at the insertion site that includes approx 30 copies of the transgene, an inversion, three duplications and a deletion in a gene desert. Detection of the transgene has proven technically challenging. We have now established transient mRNA expression during eye and brain development and are testing the presence of the exogenous protein. We are characterizing the effects of the insertion on chromatin structure and accessibility as well as of VEGF gain of function in eye development. B. Identification of coloboma candidate genes by molecular characterization of gene expression during optic fissure closure. In order to take a genome-wide approach and to accumulate hypothesis-generating data, we had completed and published a gene expression study of the margins of the optic fissure (isolated by LCM) in the developing mouse at the time of closing. We are currently pursuing single gene and screening projects to test the role of candidate genes in closure of the optic fissure and in the pathogenesis of coloboma. 1. Transcriptional regulators Zfp703/Nlz1 and Zfp503/Nlz2 We have previously published two zinc-finger motif-containing genes, Nlz1 and Nlz2, important in regulating optic fissure closure in zebrafish. Nlz1 morphant fish display multiple phenotypes such as cystic kidneys and abnormalities in heart development, likely representing a syndromic form of coloboma. We generated mice carrying targeted loxp sites (floxed) to conditionally knockout Nlz1 and Nlz2. Homozygous floxed Nlz1 mice were embryonically lethal and the surviving heterozygous showed a syndromic phenotype, including coloboma. Excision of the lacZ/Neo cassette with retention of the loxp sites resulted in viable mice with wild type phenotype. These mice are being bred to lines carrying Cre driven by eye-specific promoters for further analysis of the effects of Nlz1 conditional deletion on optic fissure closure. Nlz2 knockout mice were generated by crossing with beta actin-Cre mice. Nlz2 homozygous knockout mice were perinatally lethal and have been observed as late as postnatal day (P)1. Although overall morphologically normal, they exhibited coloboma. Differential expression by RNA-Seq of RPE at E11.5 from Nlz2-/- compared to wild type suggested the involvement of signaling pathways and extracellular matrix genes that are currently being explored. Protein expression studies in mouse eye indicated that Nlz2 is expressed transiently during development in the RPE, and in developing and adult amacrine and ganglion cells. We have not yet identified Nlz2 mutations in our coloboma patient DNA samples. 2. FAT protocadherins FAT protocadherins were among the candidate gene families suggested from our LCM screen. Fat1 and Fat4 were most highly-expressed during embryonic eye development at the optic fissure margins, periocular mesenchyme and optic cup during fissure closure. Homozygous knockout/knockdown of Fat1, but not Fat4 in mouse and zebrafish, caused coloboma. This phenotype is in part novel and in addition to the well-known glomerulotubular nephropathy observed in human patients. Eye patterning defects did not seem to be the origin of coloboma in Fat1-/- mice since their eyes were approximately normal in size until the time of optic fissure closure. The rate of cell division in the developing optic cup was mildly elevated compared to wild-type and there was no obvious change in the rate of cell death. However, we detected defects in the peri-ocular mesenchyme which could explain the coloboma phenotype. Real-time PCR revealed RPE-specific changes in expression of several important cell adhesion and signaling genes. We are currently investigating the precise molecular mechanism behind FAT1 depletion-mediated coloboma using zebrafish as a model. 3. CRISPR screening for genes associated with optic fissure closure We have developed two experimental pipelines to systematically knock out using CRISPR technology and phenotypically screen in zebrafish all 164 candidate coloboma genes identified from our gene expression study in mouse. To date, we have generated zebrafish founders (F0) carrying CRISPR-induced insertion-deletions or large deletions in 67 candidate genes. Of the 47 lines screened, 20 showed eye phenotypes, including 13 lines with coloboma in the first generation (F1). While we complete our screening, we are expanding some of the lines displaying coloboma for further characteriza